A comparative multi-level toxicity assessment of carbon-based Gd-free dots and Gd-doped nanohybrids from coffee waste: hematology, biochemistry, histopathology and neurobiology study

Here, a comparative toxicity assessment of precursor carbon dots from coffee waste (cofCDs) obtained using green chemistry principles and Gd-doped nanohybrids (cofNHs) was performed using hematological, biochemical, histopathological assays in vivo (CD1 mice, intraperitoneal administration, 14 days), and neurochemical approach in vitro (rat cortex nerve terminals, synaptosomes). Serum biochemistry data revealed similar changes in cofCDs and cofNHs-treated groups, i.e. no changes in liver enzymes' activities and creatinine, but decreased urea and total protein values. Hematology data demonstrated increased lymphocytes and concomitantly decreased granulocytes in both groups, which could evidence inflammatory processes in the organism and was confirmed by liver histopathology; decreased red blood cell-associated parameters and platelet count, and increased mean platelet volume, which might indicate concerns with platelet maturation and was confirmed by spleen histopathology. So, relative safety of both cofCDs and cofNHs for kidney, liver and spleen was shown, whereas there were concerns about platelet maturation and erythropoiesis. In acute neurotoxicity study, cofCDs and cofNHs (0.01 mg/ml) did not affect the extracellular level of L-[14C]glutamate and [3H]GABA in nerve terminal preparations. Therefore, cofNHs demonstrated minimal changes in serum biochemistry and hematology assays, had no acute neurotoxicity signs, and can be considered as perspective biocompatible non-toxic theragnostic agent.


Assessment of general toxicity of cofCDs and cofNHs. Gross toxicity and body weight change.
No toxicity was observed in any group of the experimental animals. Mortality also was not observed in the control and cofCD groups, however, 1 mouse died in cofNH group at the 6th day of the study. Furthermore, all the animals except that which died demonstrated consecutive body weight gain with no statistically significant difference between the groups, which could suggest mice wellbeing and no toxicity of the tested nanoparticles if applied in described doses for 14 days (Fig. 1).
Hematological assay. According to the data shown in Fig. 2, both cofCDs and cofNHs application caused a tendency to increase in lymphocyte (LYM) percentage (p = 0.129 and p = 0.071, respectively) with concomitant decrease in neutrophilic granulocyte (GRAN) percentages (p = 0.179 and p = 0.063, respectively). These changes are usually an evidence of some specific inflammatory process in the organism, like viral or chronic bacterial infection, autoimmune disorders 40 . However, if there is no change in the absolute WBC count, we could assume rather a shift of immune response, but not a true inflammation. Increasing LYM% may take place in case of autoimmune disorders as a consequence of lymphocyte stimulation. Indeed, and CDs could cause such impact 41 . Therefore, we suggest that the observed changes might evidence some shift in immune response against the www.nature.com/scientificreports/  www.nature.com/scientificreports/ tested nanoparticles. However, we would like to notice that the observed values were still within the normal range typical for CD-1 mice 42 . Erythrocyte (RBC) count and related parameters (hemoglobin (HGB), hematocrit (HCT), and mean corpuscular hemoglobin concentration (MCHC) values) demonstrated decreased values for both cofCDs and cofNPs in a similar manner. Decreased RBC, HGB, HCT and MCHC could evidence inhibition of erythropoiesis, and Gd probably does not contribute to this process. Then, increased mean platelet volume (MPV) with concomitant decrease of platelet (PLT) count in both groups could evidence the alteration of platelets maturation process, and, again, Gd is not supposed to be the main contributor to that. Taking together, there are some evidences of inflammation and violation of erythropoiesis and platelets formation, and it looks like that carbon core is the main contributor to these processes, and Gd adds no additional toxicity to that.
Biochemical assay. According to the data presented in Fig. 3, there are no significant changes in liver enzymes' activities, which could evidence no substantial impact of both cofCDs and cofNPs on liver function 43 . Urea's decrease in cofCDs-and cofNH-treated groups together with a tend to decrease in total protein (p = 0.147 and p = 0.129, respectively). Such data might evidence protein deficiency in the organism due to poor protein intake or malabsorption, or some problems with protein synthesis and metabolism, which both take place in liver 44 . As the mice from all the groups continuously gained weight throughout the study (Fig. 1), therefore, malnutrition can be excluded. So, the reason of urea decrease in serum might be a consequence of impaired protein metabolism. It should be noted, however, that despite the significant changes compared to control, the values of serum urea were within the normal range, typical for CD-1 mice 42 .
Absence of serum creatinine concentration changes could evidence no impact on kidney function. Taking together, both cofCDs and cofNPs might lead to inhibition of protein synthesis, but these nanoparticles in applied doses caused no substantial violation of liver and kidney functions.
Histopathology assay. According to histopathological data presented in Tables 1, 2 and 3 and in Fig. 4, both cofCDs and cofNHs did not affect substantially liver, kidney and spleen states by that way which could be considered as the injury. However, there were still some structural changes. Thus, both cofCDs and cofNHs induced slight inflammatory signs in liver, manifested by slight Kupffer cell accumulation throughout the tissue, and occasional leukocytes accumulation loci, which is in line with our hematological findings. In cofNH-treated group blood vessel congestion sometimes also took place.  www.nature.com/scientificreports/ Table 1. Pathological changes of kidney of control mice and those treated with cofCDs and cofNHs. Trait intensity: "−"-not observed, "+"-single or slight, "++"-moderate, "+++"-severe.  Table 2. Pathological changes of liver of control mice and those treated with cofCDs and cofNHs. Trait intensity: "−"-not observed, "+"-single or slight, "++"-moderate, "+++"-severe.  Table 3. Pathological changes of spleen of control mice and those treated with cofCDs and cofNHs. Trait intensity: "−"-not observed, "+"-single or slight, "++"-moderate, "+++"-severe. www.nature.com/scientificreports/ In kidneys, slight tubular epithelium loss of brush border was observed in both cofCD-and cofNH-treated groups, which, however, didn't impact on kidney function as evidenced by serum creatinine and urea levels. Also, slight tubular hyperplasia took place in these two groups, which, despite occurring during chronic progressive nephropathy, is considered as the sign of tubular cells proliferation and tubule regeneration 45 . So, it could be concluded that kidneys were affected by tested compounds, but were regenerating successfully. Then, in cofNH-treated group blood vessel dilation sometimes took place, which is similar to the finding in liver, and could evidence some alteration of these organs' blood supply.

Pathological changes Control cofCDs cofNHs
Histopathological changes in spleen were similar in both cofCD-and cofNP-treated mice. Marginal zone hyperplasia (slight) and megakaryocytosis as increased number of megakaryocytes (from slight to moderate) were observed, as well as occasional necrotic loci. Marginal zone hyperplasia might evidence some activation of phagocytic system because of being populated by macrophages predominantly 45 . Our data about potential nanoparticle accumulation in spleen are in line with the literature 46,47 . Megakaryocytosis is common in case of violation of platelet maturation and differentiation 48 , which confirms our suggestion based on hematological findings.
So, relative safety of both cofCDs and cofNHs for kidney, liver and spleen could be suggested. However, there were some concerns about platelet maturation and erythropoiesis, as well as slight inflammatory process in liver caused predominantly by carbon core. Nevertheless, these changes were minimal, so might not be an obstacle for multiple either cofCDs or cofNHs applications. Then, Gd strong capturing by cofCD core might be concluded because of no differences in biochemical, hematological and histopathological values in cofCD-and cofNH-treated groups.
Assessment of acute neurotoxicity of cofCDs and cofNHs. Fluorimetric measurements of the membrane potential of nerve terminals after application of cofCDs and cofNHs. The membrane potential was monitored using the potential-sensitive fluorescent dye rhodamine 6G. F st , the membrane potential index at the steady state level, was achieved for 5 min, and it was set as 100% in statistical calculations. In fluorimetric experiments shown in Fig. 5a,b it was revealed that both cofCDs and cofNHs did not change the membrane potential www.nature.com/scientificreports/ of nerve terminals, and so did not depolarize their plasma membrane. To compare, the time-course of KCl (35 mM)-induced membrane depolarisation of nerve terminals was presented in Fig. 5.

The extracellular level of L-[ 14 C]glutamate in nerve terminal preparations after application of cofCDs and
cofNHs. Na + -dependent glutamate and GABA transporters are strategic players in the synaptic neurotransmission mediating neurotransmitter uptake to the cytoplasm of the presynaptic nerve terminals and establishing of the proper extracellular level of the neurotransmitters. The latter is a crucial synaptic parameter that represents a dynamic energy-dependent balance between the values of the transporter-mediated uptake and unstimulated leakage of the neurotransmitters 49,50 . As shown in Table 4, the extracellular level of L-[ 14 C]glutamate in nerve terminal preparations was not changed by cofCDs and cofNHs at a concentration of 0.01 mg/ml. Table 5, the extracellular level of [ 3 H]GABA in nerve terminal preparations was not changed sig-  Table 4. The extracellular level of L-[ 14 C]glutamate in nerve terminal preparations after the application of cofCDs and cofNHs. Data were analysed using one-way ANOVA. Data are the mean ± SEM., n.s., no significant differences compared to the control; n = 12.

Nanoparticles
The extracellular level of L-[ 14

Discussion
Here, a comparative assessment of general toxicity and acute neurotoxicity of cofCDs and cofNHs was performed in vivo and in vitro experiments, respectively. Serum biochemistry results revealed no changes in main liver enzymes, which could evidence no substantial toxicity against this organ, however, some inflammatory sings were observed. Decreased urea in both groups together with a tendency to decrease of TP could evidence protein synthesis inhibition. Taking together, both compounds in applied doses caused no substantial violation of liver and kidney functions. Hematology data demonstrated increased LYM% and concomitant decreased GRAN% in both groups that could evidence some inflammatory process in the organism and confirmed our histopathological findings in liver. Decreased (significant or as a tend) RBC, HGB, HCT and MCHC in cofCDand cofNH-groups could evidence inhibition of erythropoiesis, and Gd barely contributed to that; increased MPV with concomitant decrease of PLT could evidence the alteration of platelets maturation process, which is in line with histopathological findings in spleen. Taking together, there were some evidences of inflammation and violation of erythropoiesis and platelets formation. As almost no differences in biochemical, hematological and histopathological values in cofNH-treated group were observed compared to cofCD-treated one, we could conclude that cofCD core likely contributes a lot to these processes. Our experimental data on general toxicity of cofNHs are in accordance with the literature data. In particular, the potential toxicity of Gd-CDs and Gd-DTPA (widely used contrasting agent) was compared in mice injected with Gd-CDs (Gd concentration of 5 mg/kg) and Gd-DTPA via tail vein. In blood serum biochemistry analysis, the blood samples were collected during 1-21-day period. It was revealed that Gd-CDs exhibited similar effect as compared to Gd-DTPA. Analysis of the kidney indicators, blood urea nitrogen and creatinine did not reveal difference between the control and Gd-CDs group, thereby demonstrating absence of damage to the renal function. In hepatic function analysis, AST and ALT values were slightly increased after 1-day injection. Hepatic indicators, albumin and total protein, were maintained at the normal level. Gd-CDs and Gd-DTPA may affect the hepatic function shortly after the injection without significant damage of the hepatic tissues, because the liver could quickly recover its function after 7-day injection. Taking together, these results demonstrated a low toxicity of Gd-CDs and Gd-DTPA to the animals 34 . In other study, hemolysis was not revealed after administration of Gd-CDs (prepared using hydrothermal method with 3,4-dihydroxyhydrocinnamic acid, 2,2′-(ethylenedioxy) bis(ethylamine) and Gd chloride), thereby showing little damage to red blood cells and biocompatibility with blood. Histological analysis of the organs after 24 h post injection of Gd-CDs in toxicity study in vivo revealed no obvious damages in the Gd-CDs treated groups, such as inflammatory response, pulmonary fibrosis, necrosis or damages in major organs. Long term toxicity in vivo on healthy Kunming mice model for 16 days revealed that Gd-CDs did not induce obvious hepatic or kidney disorder in mice basing on the evaluation of TP, ALT, AST, ALP, blood urea nitrogen, total cholesterol, and triglyceride. Histopathology of treated with Gd-CDs mice revealed no apparent histopathological abnormalities or lesions observed in the heart, kidney, liver, and spleen, compared to the control. Any signs of necrosis were not revealed in the histological samples. In the lung tissues, peribronchial and perivascular cellular infiltrates were demonstrated that indicated moderate lung inflammatory responses. It was concluded that Gd-CDs exhibited good biocompatibility in vivo and can be suitable for further bioapplication 31 . No hemolysis phenomenon was observed studying the hemocompatibility of Gd-CDs conjugated with AS1411 aptamers (AS1411-Gd-CDs). It has been concluded that AS1411-Gd-CDs possessed the wonderful biocompatibility as for the application in biological field 35 .
In acute neurotoxicity study in vitro, cofCDs did not affect the ambient levels of L-[ 14 C]glutamate and [ 3 H] GABA in nerve terminal preparations at a concentration of 0.01 mg/ml. CofNHs did not affect the extracellular  27 . Sulfur-containing CDs from thiourea and citric acid also had no effects on the synaptosome extracellular level of both neurotransmitters 28 . Acute neurotoxicity data in vitro on CofNHs are in accordance with the literature data. In the cytotoxicity study in vitro using human embryonic kidneys cells (293 T cells) and CCK-8 assay, Gd-CDs (prepared using hydrothermal method with 3,4-dihydroxyhydrocinnamic acid, 2,2′-(ethylenedioxy)bis(ethylamine) and Gd chloride) even at a high concentration of 1 mg/mL did not change the cell viability after 24 h incubation that indicated their low cytotoxicity in vitro 31 . In other study, it was shown that Gd-CDs obtained by a one-step hydrothermal method had the inconspicuous cytotoxicity 32 . In particular, using NIH3T3 and 4T1 cells and CCK-8 assay, it was shown that the cells preserved high viability after 24 h coincubation, thereby indicating that Gd-CDs conjugated with AS1411 aptamers induced negligible toxicity 35 .
In perspectives, we plan to optimize the methodological approaches and perform acute neurotoxicity study at significantly higher (by 50 times) cofCD and cofNH concentrations to confirm or not whether tendency to increase in the extracellular [ 3 H]GABA level in nerve terminal preparations by cofNHs (Table 4) resulted in significant increase in this parameter. This is so because a relatively low concentration of cofCDs and cofNHs was applied in vitro experiments (as compared to our previous CD-related study 27,28 ) due to untransparency and brown color of these nanoparticles. Nanoparticle-related concentrations of Gd 3+ ions (Table 5) applied to the nerve terminals were also not high. Nevertheless, these concentrations of nanoparticles interrelated to cofCD and cofNH concentrations used in vivo animal study, and are applicable for potential MRI imaging in animals. Possible capability of cofNHs to increase the extracellular [ 3 H]GABA level in nerve terminal preparations can be used as additional neurochemical theranostic feature. Theoretically, a compound that does not affect ambient glutamate level but increased the ambient GABA level in nerve terminals may possess antiepileptic, sedative and hypnotic effects.

Synthesis of cofCDs and cofNHs.
Microwave-assisted "green" synthesis of cofCDs and cofNHs from coffee waste was carried out according to a technique similar to that described in the study 51 with additional purification stages. In particular, 5 g of coffee grouts were soaked with 0.1 M GdCl 3 solution, dried, soaked with 10% NH 4 OH solution and sintered for 10 min in microwave oven in 250 ml round-bottom flask on air, making it possible to simultaneously proceed for ammoxidation reactions and interaction of hydrolyzed fragments with each other thanks to Maillard reactions, aldol condensation, alkylation of phenols, dehydration of the carbohydrates, etc. Gadolinium atoms may be retained by carboxylic groups as well as by hydroxyl groups of hydroxycinnamic acid derivatives abundant in coffee 52,53 . Then, the nanoparticles were resuspended in distilled water, filtered through Vivaspin 20 ® concentrators with polyethersulfone (PES) membranes of different pore sizes to get fraction lower than 30 kDa molecular weight, dialyzed through membrane of 3,500 MWCO (ZelluTrans ROTH ® Regenerated Cellulose Tubular Membrane), and preconcentrated using Vivaspin 20 ® of 3,000 MWCO to get nanoparticles in range 3-30 kDa. Their physical and chemical properties were partially characterized 54 , as wells as TEM images (see Fig. S1) and FTIR spectra (Fig. S2) are provided in Supplementary Information.

Ethics.
In vivo study using animals. Female CD1 mice, 10-11 weeks old, with initial body weight of 19.6 ± 3.0 g were used in the study. Animals were kept in the animal facility of the Taras Shevchenko National University of Kyiv under natural lightning at 20-23 °C, and free access to standardized rodent diet and tap water. All experiments were conducted in compliance with bioethics principles, legislative norms and provisions of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes 55 , General Ethical Principles for Experiments on Animals, adopted by the First National Bioethics Congress (Kyiv, 2001), and approved by the Institutional Animal Care and Use Committee (Protocol #1, June 24, 2021).
In vitro study using animals. Animals (Wistar rats, males, 12 weeks old, body weight of appx 120 g) were kept in the animal facilities of the Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, provided ad libitum with water and standardized rodent diet, and housed in a temperature-controlled room at 22-23 °C. Animal experiments were performed in accordance with the Guidelines of the European Community (2010/63/ EU) and local laws/policies, and were approved by the Animal Care and Use Committee of the Palladin Institute of Biochemistry (Protocol # 1 from September 21, 2020). All animal studies were reported in accordance to the ARRIVE guidelines for reporting experiments involving animals 56,57 . The total number of rats used in the study Toxicity study in vivo. Design of the study. Mice were randomly assigned onto 3 treatment groups (n = 5 in each) and received cofCDs and cofNHs dissolved in phosphate buffered saline (PBS) in concentration 25.0 mg/ml, or pure PBS (control group) intraperitoneally at the volume of 5 ml/kg (which corresponds to cof-CDs and cofNHs doses of 125 mg/kg each) daily during 14 consecutive days, according to the recommendations for repeated dose toxicity studies for preclinical drug development 58,59 . At the 15th day of the study, mice were anesthetized by 2,2,2-tribromoethanol (250 mg/kg) and sacrificed by cervical dislocation.
Examinations and observations. Mice general condition and body weight were monitored daily. The external state of the skin and fur, eyes, mucous membranes, the respiratory system, posture, and changes in spontaneous activity were evaluated. The detailed scoring system is presented in Table 7. Observations were performed immediately after the first administration, and once a day during the observation period.
Hematological assays. The blood for hematological analysis was collected immediately after the sacrifice by cardiac puncture, 25 µl of fresh blood was transferred into tubes with equal volume of 0.4% K 2 EDTA solution in saline. Assessment of the hematological parameters was performed using the hematology analyzer MCL-3124 (Guangzhou Mecan Trading Co., Ltd, China) and consumable reagents Cormay (Poland) within two hours after blood drawing. White blood cells count (WBC), lymphocyte (LYM), medium-sized cells (monocytes, eosinophils, and basophils, MID), neutrophils (GRAN) absolute and relative values, erythrocytes count (RBC), hemoglobin (HGB), hematocrit (HCT), platelet count (PLT) and average volume (MPV) were measured.
Biochemical assays. The blood for biochemical analysis was collected immediately after the sacrifice by cardiac puncture, left for 60 min to form a fibrin clot, and then centrifuged at 5400 g for 20 min at 4 °C. Blood serum was collected and used immediately for determination of the value of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), urea, creatinine, and total protein (TP). Analyses were performed on fully auto chemistry analyzer MF-240 (MedFuture LLC, USA) using standard reagent kits (Cormay, Poland) according to the protocols provided by manufacturer.
Histological assays. Liver, kidney, and spleen samples were harvested immediately after the sacrifice and fixed in 10% neutral buffered formalin for 7 days. After formalin fixation, the samples were dehydrated in ethanol solutions and embedded in paraffin, cut to obtain the slides of 5 µm thickness, which were deparaffinated and stained with hematoxylin and eosin (H&E) according to standard methods 60 , and examined under the light microscope by pathologist who was unaware of the treatment groups. Pathological features were assessed in a semi-quantitative manner, the detailed scoring systems are presented in Table 8.

Acute neurotoxicity study in vitro.
Isolation of nerve terminals (synaptosomes) from the cortex of the rat brains. The cortex brain region isolated from decapitated rats was immediately removed, and then homogenized in the ice-cold saline solution containing 0.32 M sucrose, 5 mM HEPES-NaOH, pH 7.4, and 0.2 mM EDTA. One synaptosome preparation was obtained from one rat, and each measurement was done in triplicate. www.nature.com/scientificreports/ The synaptosome preparations were obtained using differential and Ficoll-400 density gradient centrifugations of rat brain homogenate according to [61][62][63]   The synaptosome membrane potential (Em). The membrane potential of synaptosomes in the presence of cof-CDs and cofNHs was measured using the potentiometric fluorescent dye rhodamine 6G (0.5 µM) based on its potential-dependent binding to the membranes [68][69][70] . The synaptosome suspension (a final concentration of 0.2 mg of protein /ml) were preincubated at 37 °C for 10 min, and then added to a thermostated cuvette with continuous stirring. The synaptosome suspension was equilibrated with the probe, and the aliquots of cofCDs and cofNHs were added. To estimate changes in the plasma membrane potential the ratio (F) as an index of membrane potential was calculated according to Eq.: F = F t /F 0 , where F 0 and F t are fluorescence intensities of a Table 8. Organs' toxicity scale*. *Trait intensity score: "−"-not observed or less than 10%; "+"-less than 50%; "++"-less than 80%; "+++"-more than 80% of field of view/number if counted. **"−"-not observed; "+"-small occasional, "++"-small frequent, "+++"-large occasional, "++++"-large frequent. Score results are presented below in the Result section. www.nature.com/scientificreports/ fluorescent dye in the absence and presence of the synaptosomes, respectively. F 0 was calculated by extrapolation of exponential decay function to t = 0. Fluorescence measurements with rhodamine 6G were carried using a Hitachi MPF-4 spectrofluorimeter at 528 nm (excitation) and 551 nm (emission) wavelengths (slit bands 5 nm each).
Statistical analysis. GraphPad Prism 9.0.0 software was used for statistical analysis and data visualisation. Homogeneity of variance was assessed using the Levene test. The experimental data were expressed as the mean ± S.E.M. of n independent experiments. The difference between two groups was compared by one-way analysis of variance (ANOVA) with the Tukey post hoc test. Mann-Whitney U-test for independent samples was used for analysis of histopathological signs scores. Differences were considered significant, when p < 0.05.

Limitations of the study
Short-term toxicity study (14 days of administration) was performed, which does not allow to make a strict conclusion about no delayed toxicity of the tested chemicals. However, as the main purpose of Gd-dopped nanomaterials is bioimaging application, i.e. single administration, the terms used in this toxicity study allow at least to exclude acute toxicity of the nanoparticles, and therefore could be a basis for conducting preclinical animal research of these chemicals during long-term administration.

Conclusions
Summarizing, a comparative multi-level toxicity assessment showed relative safety of both cofCDs and cofNHs for kidney, liver and spleen. There were some concerns about platelet maturation and erythropoiesis, as well as potential affection of liver, but Gd incorporation was unlikely related to that. In total, cofNHs have demonstrated minimal changes in serum biochemistry and hematology assays that are not an obstacle for biomedical application of cofNHs. Also, cofCDs and cofNHs did not influence the extracellular levels of L-[ 14 C]glutamate and [ 3 H]GABA in nerve terminal preparations, and so had no acute neurotoxicity signs. Taking together, it may be considered that cofNHs can be further analyzed in biomedical research as perspective theragnostic agent.

Data availability
The datasets used during the current study are available from the corresponding author upon reasonable request. Partially, data on nanoparticle synthesis are available in the Proceedings of the C'Nano 2023: The Nanoscience Meeting; Poitiers, 2023; p. 24.